The invention relates to an apparatus and to a method for the liquid chromatographic separation of substances under pressure.
So-called chromatographic separation installations are used for the preparative and analytical separation of substance mixtures. Essentially, these installations consist in each case of a conveying unit (pump), an injection system, the actual separating device (column) and a detector. The separation of mixtures of organic components is dominated at the present time by high-pressure liquid chromatography. The reasons lie essentially in the wide range of applications and in the universality, as well as in the robustness and user friendliness of the method. It is possible to separate and detect practically any mixture of organic substances by means of high-pressure liquid chromatography. Aside from the analysis of individual samples, for which it must be possible to vary the separation parameters optimally and appropriately, there is an increasing tendency in many areas to analyze and purify large series of samples under exactly the same conditions. An exact comparability of the chromatograms and an unambiguous identification of separated substances by means of the retention times in the chromatogram are frequently needed, especially for the analytical requirements. However, unavoidable differences in the way in which chromatographic columns are filled with stationary phase material, such as the height to which the columns are filled or the packing density, can however lead to different retention times, so that an exact comparability of the chromatograms is no longer given.
Until now, for analytical and preparative purposes, individual chromatographic separation installations are used for separating individual substance mixtures. The search for pharmaceutically usable natural products and the synthesis of while libraries of substances by means of combinatory chemistry, however, had led to more stringent requirements for the sample throughput in liquid chromatographic installations in recent times.
For example, as is well known, it is possible to process sample series consecutively by serial analyses or purification of samples. However, this procedure is very time consuming and leads to long periods of time between the processing of the first and last samples. It is a disadvantage that, in carrying out liquid chromatographic separations over longer periods of time, the constancy of the conditions cannot be guaranteed, since samples, column materials and solvents, for example, may change.
Therefore, in order to analyze a large number of samples by the so-called high throughput screening, it is desirable to be able to carry out a larger number of separations simultaneously. Present parallelized separation installations require a pumping device per separating equipment (column). As a rule, however, this is uneconomic. Moreover, the individual conveying lines of such multi-channel installations exhibit retention times, which deviate from one another.
High pressure chromatographic installations are known, for which, with a total of seven pumps, one column carousel with sixteen columns, four individual detectors and one fraction collector, a maximum of four samples of four samples can be processed in parallel (Laborpraxis, December 1967, pp 61-63). In addition, because of their expensive construction in comparison to the small number of samples, which can be processed, it is not possible to work economically.
A further installation is known, with which the maximum number of samples, which can be processed in parallel, also is four (Laboratory Automation News, Vol. 2, No. 2, May 1997). Four pumps operate four columns here. Substances are detected in a UV detector, which has one deuterium lamp and four flow cells, at only two wavelengths, which can be set before the analysis. The peak recognition in the detector switches four fraction collectors. In principle, essentially several high-pressure liquid chromatography setups are used in parallel here. This is disadvantageously uneconomic.
A significant increase in the number of pumping lines can be achieved, if several channels are supplied in a parallel operation by a single pump or pump system, pumping at a constant rate, and a flow distribution, specified by the user, results.
However, because of the different flow relationships in the individual columns, a simple, uncontrolled parallel connection of several separating columns, which are supplied by a single pump, leads to a flow distribution, which can be predicted only with difficulty because of the different flow conditions in the individual columns. Before it is started up, each column must be measured for its flow properties and a characteristic flow resistance value must be obtained.
Similarly to a parallel resistance network in electric technology, one would be able to expect here also, with such a characteristic value, a corresponding distribution of the volume flow. This method of adjusting the flow in parallel operation cannot be used in practice, since it does not take into consideration any changes with time, such as aging and blocking processed in the column material.
In the DE 115 45 423 A1, an apparatus is described, with which up to 72 parallel separations are said to be possible. The apparatus is based on two circular and disk-shaped separating phases, which are connected with one another. The flow of the mobile phase is reversed in the case of this apparatus. For parallel measurements, the disks are to be provided with impermeable partitions. The detection is to be accomplished in a multi-channel detector, the details of which are not described. The separation phase is supplied by two pumps and a valve tree with mobile phase and samples. This apparatus has two critical points:    There is no detailed description of how the flows in the different channels are to be controlled when the separating columns are operated in parallel. For example, if one channel becomes blocked in the apparatus shown, the flow in the other channels, in the absence of a control system, would increase.    Likewise, it is doubtful whether the partitions on the disks prove to be tight at higher pressures. Mixing of different samples can therefore not be excluded here.